Volume 18, Issue 5, Pages 593-603 (May 2003) Herpes Simplex Virus-Specific Memory CD8+ T Cells Are Selectively Activated and Retained in Latently Infected Sensory Ganglia  Kamal M. Khanna, Robert H. Bonneau, Paul R. Kinchington, Robert L. Hendricks  Immunity  Volume 18, Issue 5, Pages 593-603 (May 2003) DOI: 10.1016/S1074-7613(03)00112-2

Figure 1 Infiltration of CD8+ T Cells in the TG after HSV-1 Corneal Infection Single-cell suspensions of TG obtained from mice 8, 14, 34, and 84 days after HSV-1 corneal infection were simultaneously stained for CD45 and CD8. For each reaction the equivalent number of cells from two TGs were stained and a total of 5 × 105 events were collected (approximately 80% of the sample). Forward and side scatter gates were set to encompass the CD45 population, and the frequency of CD8+ T cells was expressed as a percentage of CD45+ cells (% CD8). These data are representative of two to four independent experiments. Immunity 2003 18, 593-603DOI: (10.1016/S1074-7613(03)00112-2)

Figure 2 Selective Retention of CD8+ T Cells in Latently Infected Tissue TG were excised 34 days after HSV-1 corneal infection, and frozen sections were stained for CD8. Representative fields from the ophthalmic (A) and maxillary (B) branches of the TG are shown. Superimposed fluorescence and DIC images show preferential accumulation of CD8+ T cells (red) among the neuronal cell bodies in the ophthalmic branch of the latently infected TG. Immunity 2003 18, 593-603DOI: (10.1016/S1074-7613(03)00112-2)

Figure 3 Activation Phenotype of CD8+ T Cells Present in the TG Single cell suspensions of TG were analyzed for expression of (A) CD44, (B) CD69, (C) CD8 (dashed lines), or isotype control (dark line). Following flow cytometric analysis, the forward angle and side scatter gates were set on the CD45+ population. Backgating on the CD8+ population determined the proportion of CD8+ T cells that expressed the activation markers. These data are representative of two to four independent experiments. Immunity 2003 18, 593-603DOI: (10.1016/S1074-7613(03)00112-2)

Figure 4 CD8+ T Cells Retained in the TG during Latency Are Specific for HSV-1 Protein gB and Produce IFNγ Directly Ex Vivo Single cell suspensions of TG obtained (A) 14 days or (B) 34 days pi were incubated with the indicated stimulator cells for 6 hr in the presence of GolgiPlug and stained for intracellular IFNγ. The stimulator cells were HSV infected (HSV stimulated), gB498-505 peptide pulsed (gB stimulated), RR1 pulsed (RR1 stimulated), or uninfected (unstimulated). Alternatively, single cell suspensions of TGs were stained with an anti-CD8 mAb, anti-CD45 mAb, and either gB498-505/Kb or RR1822-829/Kb tetramers. A total of 5 × 105 events were collected. The dot plots represent the CD8 gated population. These data are representative of two to four experiments. Immunity 2003 18, 593-603DOI: (10.1016/S1074-7613(03)00112-2)

Figure 5 In Situ Tetramer Strain of Latently Infected TG TG were excised and gB498-505-specific CD8+ T cells were identified by simultaneously staining the whole tissue with MHC class I tetramers containing the gB498-505 epitope (green) and CD8 (red). The tissues were examined by confocal microscopy and presented as a merged image of a Z series. Areas of overlap between the CD8 and tetramer-bound TCR appear yellow. (A–C) The majority of CD8+ T cells that localized to the area of neuron cell bodies in the ophthalmic branch of the TG were tetramer positive. (D–F) Most of the CD8+ T cells exhibited ring- or patchy-foci of TCR polarization. (G) Grayscale image showing CD8+ T cells (arrows) in direct apposition to neurons (arrowheads); taking advantage of the intrinsic autofluorescence of neurons when excited with a argon laser and collected with a 500 longpass filter. RR1 tetramer was used as negative control. (H–J) Note the lack of RR1 tetramer staining on CD8 T cells in the ganglion. See supplemental data at http://www.immunity.com/cgi/content/full/18/5/593/DC1 for 3-D reconstruction. Immunity 2003 18, 593-603DOI: (10.1016/S1074-7613(03)00112-2)

Figure 6 A CD8+ T Cell Clone Specific for the gB498-505 Epitope (2D5) Can Block HSV-1 Reactivation from Latency in TG Cultures (A) TG were excised from C57BL/6 mice 34 days after corneal infection and TG cells (0.5 TG equivalent/culture) were cultured with 1 × 105 (open rectangles) 5 × 104 (hatched rectangles), or 1 × 104 (gray rectangles) 2D5 cells (n = 10), or with 1 × 105 naive CD8+ T cells (filled rectangles) (n = 5). BALB/c TG cultures were similarly prepared and incubated with 1 × 105 2D5 cells (rectangle with line) (n = 8). (B) TG were excised from C57BL/6 mice, depleted of endogenous CD8+ T cells, and cultured with 1 × 105 (n = 15) (open rectangles) or 1 × 104 (n = 20) (gray rectangles) 2D5 cells, or with 1 × 105 naive CD8+ T cells (n = 16) (filled rectangles). At the indicated times, cultures were examined and HSV-1 reactivation was monitored as described in Experimental Procedures. A Survival Curve Analysis determined the significance of differences in reactivation frequency (compared to cultures receiving naive CD8+ T cells). (*** p < 0.0001), (**p < 0.001). The reactivation frequency in cultures that received naive CD8+ T cells was not significantly different (p = 0.5485) from that in cultures to which no CD8+ T cells were added (not shown). (C) Ten days after culture initiation, total RNA was extracted from CD8+ T cell-depleted TG cultures that received 1 × 105 2D5 cells and was analyzed for HSV-1 gH transcripts by RT-PCR as described in Experimental Procedures. Each sample is comprised of pooled RNA from two cultures. Lanes 1–4 represent cultures that did not receive anti-CD8 mAb and showed no viral CPE or infectious virus. Lanes 5–8 represent cultures that received anti-CD8 mAb on day 6 of culture and were positive for viral CPE and infectious virus. Note that the cultures represented in columns 7 and 8 were nearly destroyed by the virus, resulting in greatly reduced total RNA and gH transcripts. RNA extracted from a freshly excised TG obtained 5 days after corneal infection (during lytic virus infection) served as a positive control (lanes 9 and 10). Transcripts for the housekeeping gene HPRT were similarly amplified to demonstrate equal loading of RNA from each sample. Immunity 2003 18, 593-603DOI: (10.1016/S1074-7613(03)00112-2)

Figure 7 IFN-γ Production in TG Cultures At the indicated times, supernatant fluids from the cultures described in Figure 4 were tested for IFN-γ content by ELISA. Data are presented for undepleted (A) and CD8+ T cell depleted (B) C57BL/6 TG cultures that received 1 × 105 2D5 cells or 1 × 105 naive CD8+ T cells. Immunity 2003 18, 593-603DOI: (10.1016/S1074-7613(03)00112-2)